Hans-Georg König

Vascular endothelial growth factor protects cultured rat hippocampal neurons against hypoxic injury via an antiexcitotoxic, caspase-independent mechanism.

The authors investigated the effect of vascular endothelial growth factor (VEGF) on hypoxic injury of cultured rat hippocampal neurons. Treatment with glutamate receptor antagonists prevented hypoxic neuron death. The same magnitude of protection was observed in cultures treated with VEGF, which also reduced excitotoxic neuron death induced directly by an exposure to N-methyl-d-aspartate. Vascular endothelial growth factor did not alter the activation of the transcription factor nuclear factor-κB during hypoxia and protected cells in a PI-3-kinase-independent manner. Vascular endothelial growth factor failed to protect against staurosporine-induced, caspase-dependent apoptosis. These data suggest that VEGF-induced protection against hypoxic injury primarily involves the inhibition of excitotoxic processes.

Active secretion of S100B from astrocytes during metabolic stress.

In patients suffering from cerebrovascular diseases and traumatic brain damage, increases in serum levels of protein S100B are positively correlated with the severity of the insult. Since high concentrations of S100B have been shown to exert neurotoxic effects, the objective of this study was to characterize the regulatory mechanisms underlying control of S100B release from astrocytes. To that end, we analyzed the kinetics and amount of S100B release in correlation with regulation of S100B gene expression in an in vitro ischemia model. Astrocyte cultures were treated with combined oxygen, serum and glucose deprivation, serum and glucose deprivation or hypoxia alone for 6, 12 and 24 h, respectively. While oxygen, serum and glucose deprivation triggered the most rapid release of S100B, serum and glucose deprivation provoked comparable levels of released S100B at the later time points. In contrast to oxygen, serum and glucose deprivation and serum and glucose deprivation, hypoxia alone elicited only marginal increases in secreted S100B. Parallel analysis of extracellular lactate dehydrogenase and the number of viable cells revealed only moderate cell death in the cultures, indicating that S100B was actively secreted during in vitro ischemia. Interestingly, S100B mRNA expression was potently downregulated after 12 and 24 h of oxygen, serum and glucose deprivation, and prolonged oxygen, serum and glucose deprivation for 48 h was associated with a significant reduction of S100B release at later time intervals, whereas lactate dehydrogenase levels remained constant. Our data suggest that secretion of S100B during the glial response to metabolic injury is an early and active process.

p75 neurotrophin receptor is required for constitutive and NGF‐induced survival signalling in PC12 cells and rat hippocampal neurones

We have previously shown that nerve growth factor (NGF)‐induced activation of nuclear factor‐κB increased neuronal expression of Bcl‐xL, an anti‐apoptotic Bcl‐2 family protein. In the present study we determined the role of the p75 neurotrophin receptor in constitutive and NGF‐induced survival signalling. Treatment of rat pheochromocytoma (PC12) cells with a blocking anti‐rat p75 antibody or inhibition of p75 expression by antisense oligonucleotides reduced constitutive and NGF‐induced bcl‐xL expression. Treatment with the blocking anti‐p75 antibody also inhibited NGF‐induced activation of the survival kinase Akt. Inhibition of phosphatidylinositol‐3‐kinase (PI3 kinase) activity or overexpression of a dominant‐negative mutant of Akt kinase inhibited NGF‐induced nuclear factor‐κB activation. Activation of Akt kinase by NGF was also observed in PC12nnr5 cells and cultured rat hippocampal neurones which both lack significant TrkA expression. Treatment of hippocampal neurones with the blocking anti‐p75 antibody inhibited constitutive and NGF‐induced Bcl‐xL expression, activation of Akt, and blocked the protective effect of NGF against excitotoxic and apoptotic injury. Our data suggest that the p75 neurotrophin receptor mediates constitutive and NGF‐induced survival signalling in PC12 cells and hippocampal neurones, and that these effects are mediated via the PI3‐kinase pathway.

S100B potently activates p65/c-Rel transcriptional complexes in hippocampal neurons : Clinical implications for the role of S100B in excitotoxic brain injury

Increased serum levels of S100B are positively correlated with multiple forms of CNS damage, such as stroke, CNS trauma and neurodegenerative diseases, but also in psychiatric disorders. However, it is currently not known whether increased serum levels of S100B reflect a neuroregenerative or neurodegenerative response. Since glutamate receptor overactivation (excitotoxicity) may contribute to neuronal pathology in psychiatric disorders, we investigated the effect of S100B on N-methyl-d-aspartate (NMDA)-induced neuronal cell death. Here we demonstrate that very low concentrations of S100B significantly protect primary rat hippocampal neurons against NMDA toxicity by activation of transcription factors of the Rel/nuclear factor kappaB (NF-kappaB) family. Further experiments suggest that i) S100B activated expression of the receptor of advanced glycation products (RAGE) gene in neurons and ii) S100B induced a unique composition of the active NF-kappaB complex consisting of the p65 and c-Rel subunits suggesting a novel mechanism for NF-kappaB activation involved in S100B-mediated neuroprotection. Our data suggest that S100B secreted during the glial response to brain injury potently activates p65/c-Rel in a RAGE-dependent manner and may exert neuroprotective and neuroregenerative effects in psychiatric disorders.

The amyloid precursor protein protects PC12 cells against endoplasmic reticulum stress-induced apoptosis

Endoplasmic reticulum (ER) stress is believed to play an important role in neurodegenerative disorders such as Alzheimers disease. In the present study, we investigated the effect of the human amyloid precursor protein (APP) on the ER stress response in PC12 cells. Tunicamycin, an inhibitor of N‐glycosylation, rapidly induced the expression of the ER‐resident chaperone Bip/grp78, a known target gene of the unfolded protein response. Prolonged treatment with tunicamycin (≥ 12 h) resulted in the activation of executioner caspases 3 and 7. Interestingly, PC12 cells overexpressing human wild‐type APP (APPwt) showed increased resistance to tunicamycin‐induced apoptosis compared with empty vector‐transfected controls. This neuroprotective effect was significantly diminished in cells expressing the Swedish mutation of APP (KM670/671NL). Similar effects were observed when ER stress was induced with brefeldin A, an inhibitor of ER‐to‐Golgi protein translocation. Of note, APP‐mediated neuroprotection was not associated with altered expression of Bip/grp78 or transcription factor C/EBP homologous protein‐10 (CHOP/GADD153), suggesting that APP acted either downstream or independently of ER‐to‐nucleus signaling. Our data indicate that APP plays an important physiological role in protecting neurons from the consequences of prolonged ER stress, and that APP mutations associated with familial Alzheimers disease may impair this protective activity.

Coincident enrichment of phosphorylated IκBα, activated IKK, and phosphorylated p65 in the axon initial segment of neurons

Phosphorylation of the inhibitory protein IκBα by the activated IκB kinase (IKK) is a crucial step in the activation of the transcription factor NF-κB. In neurons of the mammalian central nervous system, constitutive activation of NF-κB has been previously documented. The cellular compartments involved in this activation have not yet been fully identified. Here we document a striking enrichment of several molecules involved in NF-κB activation in the axon initial segment (AIS) of neurons: Phosphorylated-IκBα (pIκBα), activated IKK, and p65 phosphorylated at serine 536 were found to be enriched in the AIS in vivo as well as in vitro. Both, pIκBα and activated IKK, were associated with cytoskeletal components of the AIS. Activated IKK was associated with the membrane cytoskeleton, whereas pIκBα was sequestered to microtubules of the AIS. Colchicine-induced depolymerization of microtubules resulted in the loss of pIκBα in the AIS, demonstrating that the integrity of the axonal cytoskeleton is essential for the clustering of this NF-κB pathway component. These data provide the first evidence for a compartmentalized clustering of NF-κB pathway components in the AIS and implicate this neuronal compartment in the activation of NF-κB.

Full Length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons

BackgroundBcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM).ResultsWestern blot experiments confirmed a translocation of FL-Bid to the mitochondria during excitotoxic apoptosis that was associated with the release of cytochrome-C from mitochondria. These results were confirmed by immunofluorescence analysis of Bid translocation during excitotoxic cell death using an antibody raised against the amino acids 1–58 of mouse Bid that is not able to detect tBid. Finally, inducible overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 was sufficient to induce apoptosis in the hippocampal neuron cultures.ConclusionOur data suggest that translocation of FL-Bid is sufficient for the activation of mitochondrial cell death pathways in response to glutamate receptor overactivation.

AMP-activated protein kinase (AMPK)-induced preconditioning in primary cortical neurons involves activation of MCL-1.

Neuronal preconditioning is a phenomenon where a previous exposure to a sub‐lethal stress stimulus increases the resistance of neurons towards a second, normally lethal stress stimulus. Activation of the energy stress sensor, AMP‐activated protein kinase (AMPK) has been shown to contribute to the protective effects of ischaemic and mitochondrial uncoupling‐induced preconditioning in neurons, however, the molecular basis of AMPK‐mediated preconditioning has been less well characterized. We investigated the effect of AMPK preconditioning using 5‐aminoimidazole‐4‐carboxamide riboside (AICAR) in a model of NMDA‐mediated excitotoxic injury in primary mouse cortical neurons. Activation of AMPK with low concentrations of AICAR (0.1 mM for 2 h) induced a transient increase in AMPK phosphorylation, protecting neurons against NMDA‐induced excitotoxicity. Analysing potential targets of AMPK activation, demonstrated a marked increase in mRNA expression and protein levels of the anti‐apoptotic BCL‐2 family protein myeloid cell leukaemia sequence 1 (MCL‐1) in AICAR‐preconditioned neurons. Interestingly, over‐expression of MCL‐1 protected neurons against NMDA‐induced excitotoxicity while MCL‐1 gene silencing abolished the effect of AICAR preconditioning. Monitored intracellular Ca2+ levels during NMDA excitation revealed that MCL‐1 over‐expressing neurons exhibited improved bioenergetics and markedly reduced Ca2+ elevations, suggesting a potential mechanism through which MCL‐1 confers neuroprotection. This study identifies MCL‐1 as a key effector of AMPK‐induced preconditioning in neurons.

BH3-only proteins BIM and PUMA in the regulation of survival and neuronal differentiation of newly generated cells in the adult mouse hippocampus.

Neurogenesis persists in the adult hippocampus, where several thousand neurons are born every day. Most of the newly generated cells are eliminated by apoptosis, possibly because of their failure to integrate properly into neural networks. The BH3-only proteins Bim and Puma have been shown to mediate trophic factor withdrawal- and anoikis-induced apoptosis in various systems. We therefore determined their impact on proliferation, survival, and differentiation of adult-generated cells in the mouse hippocampus using gene-deficient mice. Wild-type, bim-, and puma-deficient mice showed similar rates of precursor cell proliferation, as evidenced by 5-bromo-2-deoxyuridine (BrdU)-incorporation. Deficiency in either bim or puma significantly increased the survival of adult-born cells in the dentate gyrus (DG) after 7 days. Consistently, we detected increased numbers of doublecortin (DCX)-positive and fewer terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelled-positive cells in the DG of bim- and puma-deficient mice. Bim and puma deficiency did not change early markers of neuronal differentiation, as evidenced by BrdU/DCX double-labelling. However, BrdU/NeuN double-labelling revealed that deficiency of bim, but not puma, accelerated the differentiation of newly generated cells into a neuronal phenotype. Our data show that Bim and Puma are prominently involved in the regulation of neuronal progenitor cell survival in the adult DG, but also suggest that Bim has an additional role in neuronal differentiation of adult-born neural precursor cells.

Fibroblast growth factor homologous factor 1 interacts with NEMO to regulate NF-κB signaling in neurons.

Summary Neuronal survival and plasticity critically depend on constitutive activity of the transcription factor nuclear factor-&kgr;B (NF-&kgr;B). We here describe a role for a small intracellular fibroblast growth factor homologue, the fibroblast growth factor homologous factor 1 (FHF1/FGF12), in the regulation of NF-&kgr;B activity in mature neurons. FHFs have previously been described to control neuronal excitability, and mutations in FHF isoforms give rise to a form of progressive spinocerebellar ataxia. Using a protein-array approach, we identified FHF1b as a novel interactor of the canonical NF-&kgr;B modulator IKK&ggr;/NEMO. Co-immunoprecipitation, pull-down and GAL4-reporter experiments, as well as proximity ligation assays, confirmed the interaction of FHF1 and NEMO and demonstrated that a major site of interaction occurred within the axon initial segment. Fhf1 gene silencing strongly activated neuronal NF-&kgr;B activity and increased neurite lengths, branching patterns and spine counts in mature cortical neurons. The effects of FHF1 on neuronal NF-&kgr;B activity and morphology required the presence of NEMO. Our results imply that FHF1 negatively regulates the constitutive NF-&kgr;B activity in neurons.